Nakamura Y, Suye S, Kira J, Tera H, Tabata I, Senda M
Department of Applied Chemistry and Biotechnology, Fukui University, Japan.
Biochim Biophys Acta. 1996 Mar 15;1289(2):221-5. doi: 10.1016/0304-4165(95)00143-3.
Polymerized NAD+ (Alg-NAD+) was prepared and its electrochemical properties were investigated. NAD+ has been covalently immobilized at the carboxyl group of alginic acid using water soluble carbodiimide (EDC) and then Alg-NAD+s of various NAD+ density were obtainable depending on NAD+ concentration in the reaction mixture. Absorbance of 260 nm of Alg-NAD+s showed that 3.4 to 17.6% of carboxyl groups of alginic acid were coupled with NAD+. The coenzyme activity of immobilized NAD+ has reached 80 to 90% on each Alg-NAD+. A cathodic peak in the cyclic voltammogram of Alg-NAD+ appeared at -1.2 V (vs. SCE) corresponding to the reduction wave of free NAD+. The anodic wave of NAD dimer was not observed in the presence of 2.0 mM methyl viologen and 5 units of diaphorase and NAD+ immobilized on the composite electrode could be reduced to the normal NADH. The ratio of apparent diffusion coefficient (Dapp.) of Alg-NAD+ and free NAD+ was evaluated from the variation of ipc with the square root of sweep rate (v 1/2). Despite the high molecular weight of Alg-NAD+, Dapp. Alg-NAD+/Dapp. free NAD+ are larger than that expected. These results indicate that electron transfer occurred effectively between each NAD+ molecule immobilized onto the polymer chain. It is also confirmed by a conjugated redox enzyme reaction with Alg-NAD+.
制备了聚合型烟酰胺腺嘌呤二核苷酸（Alg-NAD+）并研究了其电化学性质。使用水溶性碳二亚胺（EDC）将烟酰胺腺嘌呤二核苷酸共价固定在海藻酸的羧基上，然后根据反应混合物中烟酰胺腺嘌呤二核苷酸的浓度可获得不同烟酰胺腺嘌呤二核苷酸密度的Alg-NAD+。Alg-NAD+在260nm处的吸光度表明，海藻酸3.4%至17.6%的羧基与烟酰胺腺嘌呤二核苷酸偶联。固定化烟酰胺腺嘌呤二核苷酸的辅酶活性在每个Alg-NAD+上达到了80%至90%。Alg-NAD+的循环伏安图中的阴极峰出现在-1.2V（相对于饱和甘汞电极），对应于游离烟酰胺腺嘌呤二核苷酸的还原波。在存在2.0mM甲基紫精和5个单位的黄递酶的情况下，未观察到烟酰胺腺嘌呤二核苷酸二聚体的阳极波，并且固定在复合电极上的烟酰胺腺嘌呤二核苷酸可以还原为正常的烟酰胺腺嘌呤二核苷酸还原酶。根据峰电流（ipc）随扫描速率平方根（v1/2）的变化评估了Alg-NAD+和游离烟酰胺腺嘌呤二核苷酸的表观扩散系数（Dapp.）之比。尽管Alg-NAD+分子量较高，但Alg-NAD+的Dapp./游离烟酰胺腺嘌呤二核苷酸的Dapp.大于预期值。这些结果表明，固定在聚合物链上的每个烟酰胺腺嘌呤二核苷酸分子之间有效地发生了电子转移。这也通过与Alg-NAD+的共轭氧化还原酶反应得到了证实。
